Polynucleotide and method used for controlling insect invasion

ABSTRACT

An isolated polynucleotide includes: (a) a polynucleotide sequence as shown in SEQ ID NO: 1; or (b) a polynucleotide sequence having at least 15, 17, 19 or 21 contiguous nucleotides of SEQ ID NO: 1, wherein the growth of a pest of the order Coleoptera is inhibited when the pest of the order Coleoptera ingests double-stranded RNA comprising at least one strand that is complementary to the described polynucleotide sequence; or (c) any polynucleotide sequence as shown in SEQ ID NO: 3 to SEQ ID NO: 6; or (d) a polynucleotide sequence which hybridizes under stringent conditions with the polynucleotide sequence as defined in (a), (b) or (c). A plurality of target sequences are used for controlling a target gene c4506 of  Monolepta hieroglyphica , which is a pest of the order Coleoptera.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application of InternationalApplication No. PCT/CN2019/088924, filed on May 29, 2019, which claimspriority to Chinese Patent Application No. 201810618040.0, filed on Jun.15, 2018. The entire disclosures of the above applications are expresslyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of plant protection,especially crop protection. In particular, the present invention relatesto a polynucleotide and method for controlling insect invasions,especially to a method for controlling Monolepta hieroglyphica(Motschulsky) invasions by reducing or silencing the expression of atarget sequence in the Monolepta hieroglyphica (Motschulsky) body usingthe RNAi technology.

BACKGROUND

Crops are usually the targets of insect attacks. In the last fewdecades, there has been some substantive progress in developing moreeffective methods and compositions for controlling insect invasions incrops. For example, chemical pesticides, microbial pesticides, andgenetic engineering methods have been used to control pest invasions.

Chemical pesticides are relatively effective means for controlling pestinvasions. Nevertheless, the use of chemical pesticides also has manydisadvantages. Firstly, chemical pesticides are non-selective, and aspeople intend to apply chemical pesticides for controlling insects thatare harmful to a variety of crops and other plants, the chemicalpesticides also cause damage to non-target organisms, such asearthworms, due to their deficiency in selectivity. Moreover, afterapplying chemical pesticides for a period of time, the field usuallybecomes barren. Chemical pesticides will be present in the environmentpersistently, and will usually be metabolized slowly. Such a slowmetabolism results in the presence of chemical pesticide residues in thecrops and environment, which will be accumulated in the food chain,particularly in the food chain of higher carnivorous animals. Theaccumulation of these chemical pesticides results in the induction ofdiseases in higher species, for example cancers in humans. Therefore,there is a strong demand for an environmentally-friendly method forcontrolling or eradicating insect invasions in crop production, i.e., aselective, environmentally-friendly method with biodegradability, whichcan also be used well in a pest resistance management system.

In the last few decades, development of an effective method forcontrolling plant insect pests has achieved substantive progress.Chemical pesticides are very effective for eradicating plant pests;however, these pesticides also act on non-target insects, andfurthermore, chemical pesticides are present in the environmentpersistently, which not only causes irreversible environmentalpollution, but also results in the emergence of drug-resistant insects.Microbial pesticides, particularly pesticides obtained from the strainof Bacillus thuringiensis (abbreviated as Bt), play an important role inagricultural production as a substitute for chemical pesticides, andhave a certain insecticidal activity on insects including Lepidoptera,Diptera, Coleoptera, etc. Nevertheless, microbial pesticides have arelatively high requirement for the pesticide application environment,and if the environment is not suitable for the growth of thesemicroorganisms, repeated application needs to be performed duringproduction, and in some cases, repeated application cannot even achievethe purpose of controlling pests, thereby greatly increasing theproduction cost. Some transgenic plants which have enhanced resistanceto the pests can be obtained by introducing one or more genes encodingBt insecticidal proteins into the plants through genetic engineering,for example, genetically engineered maize and cotton plants capable ofproducing Cry toxins have been widely used in agricultural production inthe USA and provide the farmers with an alternative solution oftraditional pest-controlling methods. Nevertheless, the currentlydeveloped transgenic crops containing Cry toxins can only be used forpreventing and controlling a narrow range of Coleoptera pests, such ascorn rootworm and Colorado potato beetle. Nevertheless, there has beenno relevant report on the application of Cry toxins for the control ofMonolepta hieroglyphica (Motschulsky), one of the major pests of corn.In the meantime, Monolepta hieroglyphica (Motschulsky) is present aseggs in the soil through the winter, and in June of the following yearthe larvae hatched from the eggs also move actively in the soil. Withthe large-scale popularity of straw return-to-field measures in recentyears, it has been increasingly difficult year by year to controlMonolepta hieroglyphica (Motschulsky) by using chemical pesticides. Inparticular, in late July and early August when adult insects ofMonolepta hieroglyphica (Motschulsky) emerge from the ground and thecorn has grown to certain height, it is more difficult to control theadult insects by applying chemical pesticides.

RNA interference or RNAi is a method for down-regulating gene expressionin a sequence-specific manner in a cell or a whole organism environment,in which the purpose of directed interference with the expression of atarget gene can be achieved by the specific targeting selection andefficient mRNA repression. Although it is known in the art that the RNAitechnology can be used for preventing and controlling pests, as thereare numerous kinds of insects, this technology not only differs in itssignificantly different effects on distinct insects, and a key factorfor using such a technique as a measure for controlling insect invasionsfurther lies in selecting the mostly suitable target gene, i.e., a gene,the function of which is lost, thereby resulting in severe disruption ofthe essential biological processes and/or death of organisms. Therefore,the present invention achieves the control of insect invasions,particularly the control of insect invasions in a plant, by means ofdown-regulating a specific target gene in a pest.

SUMMARY

The object of the present invention is to provide a polynucleotide andmethod for controlling insect invasions, i.e., down-regulating theexpression of a target gene using the RNAi technology in a manner ofweakening the abilities of an insect to survive, grow, reproduce,colonize in a specific environment and/or invade a host, so as toachieve the control of insect invasions and damages caused thereby.

In order to achieve the above-mentioned object, the present inventionprovides the following technical solutions.

In one aspect, the present invention provides an isolatedpolynucleotide, which is selected from:

(a) a polynucleotide sequence as shown in SEQ ID NO: 1; or

(b) a polynucleotide sequence of at least 15 consecutive nucleotides ofSEQ ID NO: 1, wherein a double-stranded RNA comprising at least onestrand complementary to the polynucleotide sequence, when ingested by aninsect pest of Coleoptera, inhibits growth of the insect pest ofColeoptera; or(c) a polynucleotide sequence of at least 17 consecutive nucleotides ofSEQ ID NO: 1, wherein a double-stranded RNA comprising at least onestrand complementary to the polynucleotide sequence, when ingested by aninsect pest of Coleoptera, inhibits growth of the insect pest ofColeoptera; or(d) a polynucleotide sequence of at least 19 consecutive nucleotides ofSEQ ID NO: 1, wherein a double-stranded RNA comprising at least onestrand complementary to the polynucleotide sequence, when ingested by aninsect pest of Coleoptera, inhibits growth of the insect pest ofColeoptera; or(e) a polynucleotide sequence of at least 21 consecutive nucleotides ofSEQ ID NO: 1, wherein a double-stranded RNA comprising at least onestrand complementary to the polynucleotide sequence, when ingested by aninsect pest of Coleoptera, inhibits growth of the insect pest ofColeoptera; or(f) any one of the polynucleotide sequences as shown in SEQ ID NO: 3 toSEQ ID NO: 6; or(g) a polynucleotide sequence that is hybridized with or complementaryto the polynucleotide sequence as defined in any one of theabove-mentioned (a) to (f) under stringent conditions.

Preferably, the polynucleotide also comprises a complementary sequenceof the polynucleotide sequence.

More preferably, the polynucleotide sequence also comprises a spacersequence.

Most preferably, the spacer sequence is SEQ ID NO: 9.

Based on the above-mentioned technical solutions, the insect pest ofColeoptera is Monolepta hieroglyphica (Motschulsky).

In another aspect, the present invention provides an expressioncassette, comprising the polynucleotide sequence under regulation of aneffectively linked regulatory sequence.

In another aspect, the present invention provides a recombinant vectorcomprising the polynucleotide sequence or the expression cassette.

In another aspect, the present invention also provides use of thepolynucleotide sequence for interfering with expression of a targetsequence in an insect pest of Coleoptera or inhibiting growth of theinsect pest of Coleoptera.

In another aspect, the present invention also provides an interferingribonucleic acid, wherein the interfering ribonucleic acid acts todown-regulate expression of at least one target gene in an insect pestof Coleoptera after being ingested by the insect pest, wherein theinterfering ribonucleic acid comprises at least one silencing element,wherein the silencing element is a double-stranded RNA region comprisingcomplementary strands which have been annealed, and one strand of whichcomprises or consists of a nucleotide sequence at least partiallycomplementary to a target sequence within the target gene, and thetarget gene comprises the polynucleotide sequence.

Preferably, the silencing element comprises or consists of a sequence ofat least 15 consecutive nucleotides complementary to or at leastpartially complementary to a target fragment within the target sequence.

Preferably, the silencing element comprises or consists of a sequence ofat least 17 consecutive nucleotides complementary to or at leastpartially complementary to a target fragment within the target sequence.

Preferably, the silencing element comprises or consists of a sequence ofat least 19 consecutive nucleotides complementary to or at leastpartially complementary to a target fragment within the target sequence.

Preferably, the silencing element comprises or consists of a sequence ofat least 21 consecutive nucleotides complementary to or at leastpartially complementary to a target fragment within the target sequence.

Optionally, the interfering ribonucleic acid comprises at least twosilencing elements, each of which comprises or consists of a nucleotidesequence at least partially complementary to a target sequence withinthe target gene.

Preferably, each of the silencing elements comprises or consists of adifferent nucleotide sequence complementary to a different targetsequence.

More preferably, the different target sequence is derived solely from atarget gene or derived from a target gene different from the targetgene.

Further preferably, the target gene different from the target gene isderived from a same insect pest of Coleoptera or a different insect pestof Coleoptera.

Most preferably, the insect pest of Coleoptera is Monoleptahieroglyphica (Motschulsky).

Based on the above-mentioned technical solutions, the interferingribonucleic acid also comprises a spacer sequence.

Particularly, the spacer sequence is SEQ ID NO: 9.

In another aspect, the present invention also provides a composition forcontrolling invasion of an insect pest of Coleoptera, comprising atleast one of the interfering ribonucleic acids and at least one suitablecarrier, excipient or diluent.

Preferably, the composition comprises a host cell expressing or capableof expressing the interfering ribonucleic acid. Particularly, the hostcell is a bacterial cell.

More preferably, the composition is a solid, a liquid or a gel.Particularly, the composition is an insecticidal spray.

Optionally, the composition also comprises at least one pesticide,wherein the pesticide is a chemical pesticide, a potato tuber-specificprotein, a Bacillus thuringiensis insecticidal protein, a Xenorhabdusehlersii insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporus insecticidal protein or a Bacillus sphaericusinsecticidal protein.

In another aspect, the present invention also provides use of thecomposition for controlling invasion of an insect pest of Coleoptera forpreventing and/or controlling invasion of an insect pest of Coleoptera.

Preferably, the insect pest of Coleoptera is Monolepta hieroglyphica(Motschulsky).

In another aspect, the present invention also provides a method forcontrolling invasion of an insect pest of Coleoptera, comprisingcontacting the insect pest of Coleoptera with an effective amount of atleast one of the interfering ribonucleic acid sequences.

In another aspect, the present invention also provides a method forincreasing plant resistance to an insect pest of Coleoptera, comprisingintroducing the polynucleotide, the expression cassette, the recombinantvector, or a construct comprising the interfering ribonucleic acid, intothe plant.

In another aspect, the present invention also provides a method forproducing a plant capable of controlling an insect pest of Coleoptera,comprising introducing the polynucleotide, the expression cassette, therecombinant vector, or a construct comprising the interferingribonucleic acid, into the plant.

In another aspect, the present invention also provides a method forprotecting a plant from damage caused by an insect pest of Coleoptera,comprising introducing the polynucleotide, the expression cassette, therecombinant vector, or a construct comprising the interferingribonucleic acid, into the plant, wherein when ingested by the insectpest of Coleoptera, the plant being introduced acts to inhibit growth ofthe insect pest of Coleoptera.

Based on the above-mentioned technical solutions, the plant is soybean,wheat, barley, maize, tobacco, rice, rape, cotton or sunflowers.

The present invention comprises a method for regulating or inhibitingthe expression of one or more target genes in an insect pest ofColeoptera, the method comprising: introducing part or all of astabilized double-stranded RNA (such as dsRNA) or a modified formthereof (for example, a small interfering RNA sequence) into a cell ofan invertebrate harmful insect or an extracellular environment thereof.In the insect body, the dsRNA or siRNA enters the cell, inhibits theexpression of at least one or more target genes, and such an inhibitionresults in the weakening of the abilities of the insect to survive,grow, reproduce and invade a host.

The present invention provides an isolated and purified polynucleotidehaving a sequence as shown in SEQ ID NO: 1. The present invention alsoprovides any RNA expressed by the polynucleotide, including dsRNA. Thepresent invention further provides a stabilized double-stranded RNAmolecule for inhibiting the expression of a target sequence in a pest ofColeoptera. The stabilized double-stranded RNA comprises at least twocoding sequences, which are arranged in the sense and antisensedirections relative to at least one promoter, wherein the nucleotidesequences comprising a sense strand and an antisense strand areconnected or linked via a spacer sequence of at least about 5 to 1,000nucleotides, wherein the sense strand and antisense strand can be ofdifferent lengths, and wherein at least one of the two coding sequenceshas at least 80%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to any nucleotide sequence shown in SEQ ID NO: 1.

When being expressed as a dsRNA and administrated to a pest, thefragment can be defined as one resulting in death, and inhibited,hindered or halted feeding activity of the pest. The fragment can, forexample, comprise at least about 19, 21, 23, 25, 40, 60, 80, 100, 125 ormore consecutive nucleotides or about 19 to about 100 nucleotides, ormore, of any one or more sequences of SEQ ID NO: 1 or the complementarysequences thereof, such as SEQ ID NO: 3 to SEQ ID NO: 6. Particularlyuseful is a dsRNA sequence comprising about 19 to 300 nucleotideshomologous to the target sequence of pest. The present invention alsoprovides a RNA expressed by any of the polynucleotide sequences,including dsRNA. A sequence selected for expressing a gene inhibitor andfor expressing a RNA inhibiting a single gene or gene family in one ormore target pests can be constructed using a single sequence from one ormore target pests, or the DNA sequence can be constructed as a chimerafrom a variety of DNA sequences.

The plant in the present invention can include any propagation orreproduction material of a plant, and can also include a plant cell, aplant protoplast, a plant tissue culture, a plant callus and an intactplant cell in a plant or portions thereof, with these plant portionsbeing, for example, embryos, pollen, ovules, seeds, leaves, flowers,branches, fruits, kernels, ears, cobs, husks, stalks, roots, or roottips.

The Monolepta hieroglyphica (Motschulsky) in the present invention is aholometabolous insect of Galeruca, belonging to the Chrysomelidae familyof Coleoptera. Its eggs, larvae and pupae live in the soil, and theadults after emergence will fly out of the soil. It occurs onegeneration per year and passes the winter as diapause eggs, which arestarted to be hatched in May each year; larvae may be observed in thefield soil from May to early July; pupae may often be seen in the fieldfrom late-June to mid-July; adults after emergence are occasionallyflying into the fields of corn, soybeans or other plants to cause damagein mid-July; the peak period of emergence is from late-July to earlyAugust. The adult insects will be ready to lay eggs about 15 days afteremergence. The egg-laying period lasts for about 1 month.

The larvae of Monolepta hieroglyphica (Motschulsky) feed mainly on theroots of crops in the farm field, and thus the damage caused therebycannot be seen above the ground; it can be found in mid-July each yearthat their adults are damaging the leaves in corn and soybean fields;from late-July to early August a large number of adults are damaging thecorn silk by biting off the corn silk and severely impairingpollination, resulting in pointed and spindle-shaped ears, and thuslower corn yield. Subsequently, the Monolepta hieroglyphica(Motschulsky) adults will migrate to soybean fields to eat soybeanleaves or to the surrounding vegetable fields to damage the vegetables.The damage area of corn caused by Monolepta hieroglyphica (Motschulsky)from 2009 to 2016 in China had been doubled from 16 million mu (i.e.,about 1.07 million hectare) to 40 million mu (i.e., about 2.67 millionhectare), and the damage regions had been expanded from the Northwest tothe major corn-growing areas such as Northeast and Northern parts ofChina.

In the meantime, with the continuous advancement of strawreturn-to-field measures, field humus and substances covering the soilsurface have been increasingly enriched and accumulated, and thus it ismore difficult to apply pesticides to the soil and the control ofMonolepta hieroglyphica (Motschulsky) also becomes trickier. In otherwords, straw return-to-field, which furnishes a natural shelter for theMonolepta hieroglyphica (Motschulsky) larvae, may lead to a much highersurvival rate of Monolepta hieroglyphica (Motschulsky) larvae, therebyresulting in higher population density of the Monolepta hieroglyphica(Motschulsky) insects. The Monolepta hieroglyphica (Motschulsky) adults,as the insects which can skillfully fly and jump, will start to damagethe corn in the mid- to late-July after emergence when the corn isgrowing at silking stage. In this case, the corn has grown to certainheight; and the application of pesticides becomes more difficult and islikely to cause a sad tragedy of accidentally hurting a person who isapplying them. Also, the non-selective insecticidal effect can causedamage to crops and non-target organisms. Moreover, chemical pesticidesmay have a cumulative effect in the human body to become mutagens orcarcinogens. Therefore, there is a need to find a precise andenvironmentally friendly method that can be simply and easily operatedby a farmer to control the damage caused by Monolepta hieroglyphica(Motschulsky). By using genetic modification, the crops can have certaininsecticidal efficacy against the pests over the entire growing period,and the entire plants are protected during their whole growing period.To address the above problems, the best solution is to adopt a method ofcontrolling Monolepta hieroglyphica (Motschulsky) by using geneticallymodified RNAi means so as to provide the corn with complete-control overthe entire plants during the whole growing period.

The expression “controlling an insect” or “controlling a pest” or“controlling an insect pest” in the present invention means any effecton an insect which can result in limitation of the damage caused by theinsect, including, but not limited to, killing the insect, inhibitingdevelopment of the insect, changing fertility or growth of the insect insuch a manner that the insect can only cause less damage to the plant,reducing the quantity of progenies generated by the insect, producingless normal insects, producing insects which will be more easilyattacked by predators or preventing the insects from eating the plants.

The expression “target gene” in the present invention means any sequenceintended to be down-regulated in an insect. Insect infestations arecontrolled by down-regulating the target gene, for example by disruptingnecessary biological processes in the insects. Therefore, preferredtarget genes include, but are not limited to, genes playing essentialroles in regulating feeding activity, survival, growth, development,reproduction, invasion and infection. When the expression of the targetgene is down-regulated or inhibited, at least 30% of the insects arekilled; or the growth of at least 30% of the insects isprevented/slowed/hindered/delayed/blocked, the reproduction of at least30% of the insects is prevented, and the change in at least 30% of theinsects through their life cycle is prevented; or the damage caused bythe insects and/or the abilities of the insects to infect or infest theenvironment, surface and/or plants or crop species is decreased; or atleast 30% of the insects are stopped feeding from natural food sourcesthereof (such as a plant and a plant product). These target genes can beexpressed in all or a portion of insect cells. Additionally, thesetarget genes can be only expressed in a specific stage in a life cycleof the insects, for example in the adult stage, larval phase or eggstage.

In the present invention, the term “pest” is preferably an insectcausing plant invasion/infestation/infections, and belongs toColeoptera, preferably Monolepta hieroglyphica (Motschulsky). The terms“infestation”, “infection” and/or “invasion” can be generally usedinterchangeably throughout the document.

The term “RNA interference (RNAi)” in the present invention means someRNAs that can high efficiently and specifically block the expression ofa specific gene in vivo, promote the degradation of mRNA, and induce acell to exhibit a specific gene deletion phenotype; this technology isalso referred to as RNA intervention or interference. RNA interferenceis a highly specific gene silencing mechanism at the mRNA level.

The term “nucleic acid” in the present invention means a single-strandedor double-stranded polymer of deoxyribonucleic acid or ribonucleic acidbases read from the 5′-terminus to 3′-terminus. Optionally, the term“nucleic acid” can also comprise non-naturally occurring or changedbases which allow correct reading by a polymerase and will not reducethe expression of a polypeptide encoded by the nucleic acid. The term“nucleotide sequence” means a sense strand and an antisense strand of anucleic acid present as individual single strands or present in a dimer.The term “ribonucleic acid” (RNA) includes RNAi (RNA interference),dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA(messenger RNA), miRNA (microRNA), tRNA (transfer RNA charged with orwithout corresponding acylated amino acids) and cDNA and genomic DNA, aswell as DNA-RNA hybrids. The term “nucleic acid fragment”, “nucleic acidsequence fragment” or the more commonly-known term “fragment” will beunderstood by a person skilled in the art to include a genomic sequence,a ribosomal RNA sequence, a transfer RNA sequence, a messenger RNAsequence, an operon sequence and a smaller engineered nucleotidesequence, wherein these sequences express or can be engineered toexpress a protein, a polypeptide or a peptide.

The term “interfering ribonucleic acid” in the present invention coversany type of RNA molecule capable of down-regulating or “silencing” theexpression of a target sequence, including, but not limited to, senseRNA, antisense RNA, siRNA, miRNA, dsRNA, hairpin RNA (hpRNA), and thelike. Methods for measuring functional interfering RNA molecules arewell known in the art and have been disclosed.

The interfering ribonucleic acid in the present invention achievesspecific down-regulation of the expression of a target gene by bindingto a target sequence within the target gene. The reason for theoccurrence of the binding is the base pairing between the complementaryregions of the interfering RNA and the target sequence.

The present invention encompasses nucleic acid molecules or fragmentsthereof that are hybridized (in particular specifically hybridized) withthe polynucleotide according to the present invention under “stringentcondition”. As known to the person skilled in the art, nucleic acidmolecules or fragments thereof are capable of specifically hybridizingwith other nucleic acid molecules under certain conditions. In thepresent invention, if two nucleic acid molecules can form anantiparallel nucleic acid structure with double strands, it can bedetermined that these two molecules can hybridize with each otherspecifically. If two nucleic acid molecules are completelycomplementary, one of the two molecules is called as the “complement” ofthe other one. In this invention, when every nucleotide of a nucleicacid molecule is complementary to the corresponding nucleotide ofanother nucleic acid molecule, it is identified that the two moleculesare “completely complementary”. If two nucleic acid molecules canhybridize with each other with enough stability so that they can annealto and bind to each other under at least normal “low-stringent”conditions, these two nucleic acids are identified as “minimumcomplementary”. Similarly, if two nucleic acid molecules can hybridizewith each other with enough stability so that they can anneal to andbind to each other under normal “high-stringent” conditions, it isidentified that these two nucleic acids are “complementary”. Deviationfrom “completely complementary” can be allowed, as long as the deviationdoes not completely prevent the two molecules to form a double-strandstructure. A nucleic acid molecule which can be taken as a primer or aprobe must have sufficiently complementary sequences to form a stabledouble-strand structure in the specific solvent at a specific saltconcentration. In the present invention, basically homologous sequencerefers to a nucleic acid molecule, which can specifically hybridize withthe complementary strand of another matched nucleic acid molecule under“high-stringent” conditions. The stringent conditions for DNAhybridization are well-known to those skilled in the art, such astreatment with 6.0×sodium chloride/sodium citrate (SSC) solution atabout 45° C. and washing with 2.0×SSC at 50° C. For example, the saltconcentration in the washing step is selected from 2.0×SSC and 50° C.for the “low-stringent” conditions and 0.2×SSC and 50° C. for the“high-stringent” conditions. In addition, the temperature in the washingstep ranges from about 22° C. for the “low-stringent” conditions toabout 65° C. for the “high-stringent” conditions. Both temperature andthe salt concentration can vary together, or one of them can remainunchanged while the other variable changes. Preferably, thepolynucleotide of this invention is specifically hybridized in 6.0×SSCand 0.5% SDS solution at 65° C. for the “high-stringent” conditions;then the membrane was washed once in 2×SSC and 0.1% SDS solution and in1×SSC and 0.1% SDS solution, respectively.

The term “silencing element” refers to a part or region of aninterfering ribonucleic acid comprising or consisting of a nucleotidesequence complementary to or at least partially complementary to atarget sequence within a target gene, wherein the part or region acts asan active part of the interfering ribonucleic acid so as to direct thedown-regulation of the expression of the target gene. The silencingelement comprises a sequence having at least 15 consecutive nucleotides,preferably at least 18 or 19 consecutive nucleotides, more preferably atleast 21 consecutive nucleotides, and even more preferably at least 22,23, 24 or 25 consecutive nucleotides complementary to a target sequencewithin a target gene; or an interfering ribonucleic acid consistingthereof.

The term “expression of a target gene” in the present invention refersto the transcription and accumulation of RNA transcripts encoded by atarget gene and/or translation of mRNA into a protein.

The term “down-regulation” refers to any of the methods known in the artby which an interfering ribonucleic acid reduces the level of primaryRNA transcript, mRNA or protein produced from a target gene. Thedown-regulation refers to a situation whereby the level of RNA orproteins produced from a gene is reduced by at least 10%, preferably atleast 33%, more preferably at least 50%, and even more preferably atleast 80%. Specifically, down-regulation refers to the reduction of thelevel of RNA or proteins produced from a gene in an insect cell by atleast 80%, preferably at least 90%, more preferably at least 95%, andmost preferably at least 99%, as compared with a suitably controlledinsect (for example, an insect which has not been exposed to theinterfering ribonucleic acid or has been exposed to a controlinterfering ribonucleic acid). Methods for detecting the reduction ofRNA or protein levels are well known in the art, and include RNAsolution hybridization, Northern hybridization, reverse transcription(for example quantitative RT-PCR analysis), microarray analysis,antibody binding, enzyme-linked immunosorbent assay (ELISA) and Westernblotting. Meanwhile, down-regulation can also mean that, as comparedwith the suitable insect control, the level of RNA or proteins isreduced to a level sufficient to result in the insect phenotypegenerating a detectable change, for example cell death, growthcessation, and the like. Therefore, down-regulation can be measured byphenotype analysis of the insect using conventional techniques in theart.

The expression “inhibition of expression of a target gene” in thepresent invention refers to the reduction or absence (below a detectablethreshold) of the level of the proteins and/or mRNA product of thetarget gene. Specificity refers to an ability to inhibit a target geneand produce no effect on other genes in a cell, and brings about noeffect on any gene in a cell generating dsRNA molecules.

The “sense” RNA in the present invention refers to an RNA transcriptcorresponding to a sequence or fragment present in the form of mRNAwhich can be translated into a protein by a plant cell. The “antisense”RNA in the present invention refers to RNA complementary to all or partof mRNA produced normally in a plant. The complementation of anantisense RNA can be directed at any part of a transcript of a specificgene, i.e. a 5′ non-coding sequence, 3′ non-coding sequence, intron orcoding sequence. The term “RNA transcript” in the present inventionrefers to a product obtained by transcription catalyzed by an RNApolymerase performed on the DNA sequence. When the RNA transcript is acompletely complementary copy of a DNA sequence, the RNA transcript isreferred to as a primary transcript, or is an RNA obtained bypost-transcriptional processing of the primary transcript, which isreferred to as a mature RNA.

The interfering ribonucleic acid in the present invention down-regulatesthe expression of a gene by RNA interference or RNAi. RNAi is a typicalmethod for sequence-specific gene regulation mediated by adouble-stranded RNA molecule (such as siRNA). siRNA comprises a senseRNA strand being annealed with an antisense RNA strand by complementarybase pairing. The sense strand or “leading strand” in a siRNA moleculecomprises a nucleotide sequence complementary to a nucleotide sequencelocated within an RNA transcript of a target gene. Therefore, the sensestrand of siRNA can be annealed with the RNA transcript byWaston-Crick-type base pairing, and targets the RNA so that the RNA isdegraded in a cellular complex referred to as RNAi induced silencingcomplex or RISC. In the case of a preferred interfering ribonucleic acidin the present invention, the silencing element can be a double-strandedregion comprising complementary strands being annealed, at least onestrand of which comprises a nucleotide sequence complementary or atleast partially complementary to a target sequence within a target gene;or comprises an interfering ribonucleic acid consisting thereof. Thedouble-stranded region has a length of at least about 15 to about 25base pairs, or a length of about 25 to about 100 base pairs, or even alength of about 3,000 base pairs.

The dsRNA molecule in the present invention can serve as a precursor foractive siRNA molecules which direct RNA transcripts to the RISC complexfor subsequent degradation. A dsRNA molecule present in an organism orthe cellular surroundings thereof can be ingested by the organism andprocessed by an enzyme known as DICER to obtain a siRNA molecule.Optionally, a dsRNA molecule can be produced in vivo, i.e., one or morepolynucleotides encoding the dsRNA present in a cell (for example, abacterial cell or a plant cell) are transcribed, and processed by DICERin a host cell or preferably in an insect cell after ingesting a longerprecursor dsRNA. The dsRNA can be formed by two separate (sense andantisense) RNA strands being annealed by complementary base pairing.Alternatively, dsRNA can be a single strand, which can refold itself toform a hairpin RNA or a stem-loop structure. In the case of one singleRNA, the double-stranded region or “stem” is formed of two regions orsegments of the RNA, wherein these regions or segments are substantiallyinverted repeat sequences for each other, and have sufficientcomplementarity to allow the formation of a double-stranded region. Oneor more functional double-stranded silencing elements can be present inthis “stem region” of the molecule. Inverted repeat regions aretypically spaced via a region or segment referred to as a “loop” regionin an RNA. This region can comprise any nucleotide sequence whichconfers sufficient flexibility to allow self-pairing between flankingcomplementary regions of RNA, and in general, the loop region issubstantively single stranded and serves as a spacer sequence betweeninverted repeat sequences.

The interfering ribonucleic acid in the present invention comprises atleast one double-stranded region, typically a silencing element of theinterfering ribonucleic acid, which comprises a sense RNA strand beingannealed with an antisense RNA strand by complementary base pairing,wherein the sense strand of the dsRNA molecule comprises a nucleotidesequence complementary to a nucleotide sequence located within the RNAtranscript of a target gene. The silencing element or at least onestrand thereof (when the silencing element is double stranded) can becompletely or partially complementary to a target sequence of a targetgene. The term “completely complementary” means that all the bases ofthe nucleotide sequence of a silencing element are complementary to or“match” the bases of a target sequence. The term “at least partiallycomplementary” refers to less than 100% of matching degree being presentbetween the bases of a silencing element and the bases of a targetsequence. A person skilled in the art would understand that in order tomediate the down-regulation of the expression of a target gene, thesilencing element only needs to be at least partially complementary tothe target sequence. It is known in the art that a RNA sequence havingan insertion, deletion and mismatch with respect to the target gene canstill be effective in terms of RNAi. Preferably, the silencing elementand the target sequence of the target gene share at least 80% or 85%sequence identity, preferably at least 90% or 95% sequence identity, ormore preferably at least 97% or 98% sequence identity, and still morepreferably at least 99% sequence identity. Optionally, over each lengthof 24 partially complementary nucleotides, as compared with the targetsequence, the silencing element can comprise 1, 2 or 3 mismatches. It iswell known to a person skilled in the art that the complementaritydegree shared between the silencing element and the target sequencevaries with the expression of the target gene to be down-regulated orthe insect species to be controlled.

The target sequence in the present invention can be selected from anysuitable region or nucleotide sequence of a target gene or an RNAtranscript thereof. For example, the target sequence can be locatedwithin the 5′ UTR or 3′ UTR of the target gene or RNA transcript, orwithin an extron or intron region of the gene.

The interfering ribonucleic acid in the present invention can compriseone or more silencing elements, wherein each silencing element comprisesor consists of a nucleotide sequence at least partially complementary toa target sequence within a target gene, and functions to down-regulatethe expression of the target gene after being ingested by an insect. Theterm “a plurality of” or “more” means at least two, at least three, atleast four, and so on until at least 10, 15, 20 or at least 30. Theinterfering ribonucleic acid comprises a plurality of copies of a singlesilencing element, i.e., repeats of the silencing element binding to aspecific target sequence within a specific target gene. The silencingelement within the interfering ribonucleic acid can also comprise orconsist of different nucleotide sequences complementary to differenttarget sequences. It shall be apparent that a combination of a pluralityof copies of the same silencing element and a silencing element bindingto a different target sequence also falls within the scope of thepresent invention.

In the present invention, in order to achieve the down-regulation of aspecific target gene in an insect of Coleoptera, different targetsequences can be derived from a single target gene in an insect. In thiscase, silencing elements in an interfering ribonucleic acid can becombined according to the original order of target sequences present ina target gene, or as compared with the order of the target sequences inthe target gene, the silencing elements can be disorganized and randomlycombined in any rank order in an environment of the interferingribonucleic acid.

Optionally, different target sequences represent a single target generespectively, but are derived from different insect species.

Optionally, different target sequences can be derived from differenttarget genes. If an interfering ribonucleic acid is used for preventingand/or controlling pest invasions, then it is preferred that differenttarget sequences are selected from the group consisting of genesregulating necessary biological functions of an insect, wherein thesebiological functions include, but are not limited to, survival, growth,development, reproduction and pathogenicity. The target sequences canregulate the same or different biological pathways or processes.

In the present invention, different genes targeted by differentsilencing elements can be derived from the same insect. This method canbe used for achieving an enhanced attack against a single insect.Particularly, different target genes can be differentially expressed indifferent stages of life cycle of the insect, for example the matureadult stage, immature larval stage and egg stage. Therefore, theinterfering ribonucleic acid in the present invention can be used forpreventing and/or controlling insect invasions in one or more stages ofthe life cycle of the insect. Alternatively, different genes targeted bydifferent silencing elements are derived from different insects;therefore, the interfering ribonucleic acid in the present invention canalso be used for simultaneously preventing and/or controlling invasionsof one or more types of insects.

The silencing element in the present invention can be a consecutiveregion of an interfering ribonucleic acid or can be spaced apart via alinker sequence. The linker sequence can comprise a short randomnucleotide sequence that is not complementary to any target sequence ortarget gene. The linker sequence can be a conditional self-cleavage RNAsequence, preferably a pH sensitive linker or a hydrophobic sensitivelinker. The linker can also comprise a nucleotide sequence equivalent toan intron sequence. The length of the linker sequence can be in a rangeof 1 base pair to about 10,000 base pairs, provided that the linker willnot weaken the ability of the interfering ribonucleic acid todown-regulate the gene expression.

In addition to one or more silencing elements and any linker sequence,the interfering ribonucleic acid in the present invention can alsocomprise at least one additional polynucleotide sequence. The additionalpolynucleotide sequence is selected from: (1) a sequence capable ofprotecting the interfering ribonucleic acid from RNA processing; (2) asequence affecting the stability of the interfering ribonucleic acid;(3) a sequence which allows binding of a protein to facilitate theingestion of the interfering ribonucleic acid by an insect cell; (4) asequence facilitating the large-scale production of the interferingribonucleic acid; (5) an aptamer sequence capable of binding to anreceptor or binding to a molecule on surface of an insect cell so as tofacilitate the ingestion; or (6) a sequence catalyzing the processing ofthe interfering ribonucleic acid in an insect cell and thereby enhancingthe efficacy of the interfering ribonucleic acid.

The length of the interfering ribonucleic acid in the present inventionneeds to be sufficient to be ingested by an insect cell anddown-regulate a target gene in the insect. The upper limit of the lengthcan depend on: (1) the requirement for ingestion of the interferingribonucleic acid by an insect cell, and (2) the requirement of theinterfering ribonucleic acid in the insect cell being processed tomediate gene silence through an RNAi approach, and the length can alsobe specified by a method of production and a formulation for deliveringthe interfering ribonucleic acid to the cell. Preferably, the length ofthe interfering ribonucleic acid in the present invention will bebetween 19 and 10,000 nucleotides, preferably between 50 and 5,000nucleotides or between 100 and 2,500 nucleotides, more preferably havinga length between 80 and 2,000 nucleotides.

The interfering ribonucleic acid in the present invention can compriseDNA bases, unnatural bases or an unnatural backbone connection ormodifications of a sugar-phosphate backbone, for example, for enhancingthe stability during storage or enhancing the resistance to nucleasedegradation. Additionally, the interfering ribonucleic acid can beproduced chemically or enzymatically through a manual or automaticreaction by a person skilled in the art. Optionally, the interferingribonucleic acid can be transcribed from a polynucleotide encodingthereof. Therefore, the present invention provides an isolatedpolynucleotide for encoding any one of the interfering ribonucleic acid.

The polynucleotide in the present invention can be inserted into a DNAconstruct or a vector known in the art by a conventional molecularcloning technique. The DNA construct can be a recombinant DNA vector,for example, a bacterial, viral or yeast vector. The DNA construct is anexpression construct, in which the polynucleotide is operably linked toat least one regulatory sequence capable of driving the expression ofthe polynucleotide sequence. The term “regulatory sequence” refers toany nucleotide sequence capable of affecting the expression of anoperably linked polynucleotide, including, but not limited to, apromoter, an enhancer, and other naturally generated or synthesizedtranscriptional activation elements. The regulatory sequence can belocated at the 5′ or 3′ terminus of the polynucleotide sequence. Theterm “operably linked” refers to a functional connection between aregulatory sequence and a polynucleotide sequence, in which theconnection makes the regulatory sequence drive the expression of thepolynucleotide. Operably linked elements can be consecutive orinconsecutive.

The regulatory sequence in the present invention can be a promoter.Preferably, the promoter is a plant expressible promoter. The “plantexpressible promoter” refers to a promoter that ensures the expressionof the polynucleotide linked thereto in a plant cell. The plantexpressible promoter can be a constitutive promoter. Examples ofpromoters directing the constitutive expression in plants include, butare not limited to, a 35S promoter derived from cauliflower mosaicvirus, maize ubi promoters, rice GOS2 gene promoters, and the like.Alternatively, the plant expressible promoter can be a tissue specificpromoter, i.e. the promoter directs the expression of an coding sequencein several tissues, such as green tissues, at a level higher than inother tissues of the plant (which can be measured through conventionalRNA trials), such as a PEP carboxylase promoter. Alternatively, theplant expressible promoter can be a wound-inducible promoter. Thewound-inducible promoter or a promoter directing the expression modeinduced by the wound means that when a plant suffers from a wound causedby a mechanical factor or the gnawing of insects, the expression of thepolynucleotide under the regulation of the promoter is significantlyimproved than when under normal growth conditions. Examples of thewound-inducible promoters include, but are not limited to, promoters ofpotato and tomato protease inhibitor genes (pinI and pinII) and maizeprotease inhibitor genes (MPI).

Optionally, one or more transcription termination sequences can beincorporated into the expression construct in the present invention. Theterm “transcription termination sequence” covers a control sequence atthe terminus of a transcription unit, and sends signals regarding thetranscription termination, 3′ processing and polyadenylation of aprimary transcript. The additional regulatory element includes, but isnot limited to, a transcription or translation enhancer which can beincorporated into an expression construct, for example, a doubleenhancing CaMV35S promoter.

The method for producing any interfering ribonucleic acid in the presentinvention comprises the steps of: (1) contacting the polynucleotideencoding the interfering ribonucleic acid or a DNA construct comprisingthe polynucleotide with a cell-free component; and (2) introducing thepolynucleotide encoding the interfering ribonucleic acid or the DNAconstruct comprising the polynucleotide (for example, throughtransformation, transfection or injection) into a cell.

In the present invention, a host cell comprising any interferingribonucleic acid of the present invention, any polynucleotide of thepresent invention or a DNA construct comprising these polynucleotidescan be a prokaryotic cell, including, but not limited to, Gram-positiveand Gram-negative bacterial cells; or a eukaryotic cell, including, butare limited to, a yeast cell or a plant cell. Preferably, the host cellis a bacterial cell or a plant cell. The polynucleotide or DNA constructin the present invention can be present or maintained as anextrachromosomal element in the host cell, or can be stably incorporatedinto the genome of the host cell.

In the present invention, in the case of an interfering ribonucleic acidbeing expressed in a host cell and/or used for preventing and/orcontrolling insect infestations in a host organism, it is preferred thatthe interfering ribonucleic acid does not exhibit a significant“off-target” effect, i.e., the interfering ribonucleic acid does notaffect the expression of a non-target gene in the host. Preferably, thesilencing gene does not exhibit significant complementarity to anucleotide sequence apart from a given target sequence of the targetgene. The silencing element shows less than 30%, more preferably lessthan 20%, more preferably less than 10%, and even more preferably lessthan 5% sequence identity to any gene of the host cell or organism. Ifthe genomic sequence data of the host organism is available, then theidentity to the silencing element can be crosschecked using standardbioinformatics tools. Within a region having 17 consecutive nucleotides,more preferably within a region having 18 or 19 consecutive nucleotides,and most preferably within a region having 19 or 20 or 21 consecutivenucleotides, the silencing element and the gene from the host cell ororganism do not have sequence identity.

In the present invention, the composition for preventing and/orcontrolling insect infestations comprises at least one interferingribonucleic acid and optionally at least one suitable carrier, excipientor diluent, wherein the interfering ribonucleic acid functions todown-regulate the expression of a target gene in an insect after beingingested by the insect. The interfering ribonucleic acid comprises orconsists of at least one silencing element, and the silencing element isa double-stranded RNA region containing complementary strands beingannealed, one strand of which (sense strand) comprises a nucleotidesequence at least partially complementary to a target sequence within atarget gene. The target gene includes, but is not limited to, genesregulating the survival, growth, development, reproduction andpathogenicity of an insect. Optionally, the composition comprises atleast one host cell, and the host cell comprises at least oneinterfering ribonucleic acid or a DNA construct encoding the interferingribonucleic acid, and optionally at least one suitable carrier,excipient or diluent, wherein the interfering ribonucleic acid functionsto down-regulate the expression of a target gene in an insect after thehost cell is ingested by the insect.

The composition of the present invention can be presented as anysuitable physical form to be applied to an insect. For example, thecomposition can be in the form of a solid (powder, pellet or bait), aliquid (including an insecticidal spray) or a gel. The composition canbe a coating, paste or powder, which can be applied to a substrate so asto protect the substrate from the insect infestation. The compositioncan be used for protecting any substrate or material sensitive to theinsect invasions or damage caused by the insect.

The properties of the excipient and the physical form of the compositioncan vary due to the properties of the substrate which is desired to betreated. For example, the composition can be a liquid which is brushedor sprayed onto a material or substrate to be treated or printed onto amaterial or substrate to be treated; or a coating or powder which isapplied to a material or substrate to be treated.

In the present invention, the composition can be in the form of bait.The bait is used to induce an insect to be contacted with thecomposition. After being in contact with the insect, the composition issubsequently internalized by the insect through, for example, ingestionand mediates RNAi, thereby killing the insect. The bait can comprise atype of food, such as a type of protein-based food, for example fishmeal. Boric acid can also be used as bait. The bait can depend upon thespecies to be targeted. An attractant can also be used, which, forexample, can be a pheromone such as a male or female pheromone. Theattractant can act to induce the contact between the insect and thecomposition, and can be targeted at a specific insect or can attractinsects over the whole range, increasing the contact chance of theinduced insects and the composition of the present invention, therebyachieving the purpose of killing a mass of insects. The bait can be inany suitable form, such as the form of a solid, a paste, a pellet or apowder.

The bait can also be taken by an insect to the insect community. Thebait can then serve as a food source of other members in the community,thereby providing an effective control for a mass of insects andpotentially the whole insect community. The bait can also be provided ina suitable “shell” or “trapper”.

Additionally, the composition in contact with the insects can be held onthe surface of the insects. Upon cleaning, whether cleaning a singleinsect on its own or cleaning each other, the composition can beingested and can thus mediate the effect thereof in the insects. Forthis, the composition needs to be sufficiently stable, so that even whenexposed to external environment conditions for a period of time (forexample, several days), the interfering ribonucleic acid still remainsintact and can mediate RNAi.

The composition in the present invention can be provided in the form ofa spray. Therefore, a human user can directly spray the insects with thecomposition. The composition is then internalized by an insect, and canmediate RNA interference in the insect body, thereby controlling theinsect. The spray is preferably a pressurized/atomized spray or a pumpspray. These particles can have a suitable size so that they can beadhered to the insect, for example, adhered to the exoskeleton where theparticles can be absorbed.

In the present invention, the carrier of the composition is anelectrostatic powder or particle, which can be adhered to an insect.Optionally, the carrier of the composition can comprise magneticparticles, which can be adhered to the surface of the insect.Optionally, the carrier of the composition comprises metal particles,which are initially unmagnetized, but can become magnetically polarizedupon entering an electric field provided by the insect body. Preferably,the composition is incorporated into a carrier which increases theingestion of an interfering RNA by the insect. Such a carrier can be alipid-based carrier, preferably including one or more of the following:an oil-in-water type emulsion, a micelle, cholesterol, lipopolyamine andliposome. Other agents improving the ingestion of the construct of thepresent invention are well known to a person skilled in the art, andinclude polycations, dextran and cationic lipids such as CS096 andCS102. Optionally, the carrier of the composition is a coagulant fornucleic acid, and preferred coagulant comprises spermidine or protaminesulfate, or derivatives thereof.

In the case that the composition of the present invention is suitablefor preventing and/or controlling insect invasions in a plant, thecomposition can comprise an agriculturally suitable carrier. Such acarrier can be any material which can be tolerated by a plant to betreated, and the material would not cause inappropriate damage to theenvironment or other organisms therein, and allows the efficacy of theinterfering ribonucleic acid on the insect to be maintained. Inparticular, the composition of the present invention can be formulatedin accordance with the conventional agricultural practice used in theindustry of biological pesticides, so as to be delivered to a plant. Thecomposition can comprise an additional component capable of performingother functions, wherein these functions include, but are not limitedto, (1) enhancing or improving the ingestion of the interferingribonucleic acid by an insect cell, and (2) stabilizing the activecomponents of the composition. Such additional component contained inthe composition comprising the interfering ribonucleic acid can be ayeast tRNA or yeast total RNA.

The composition can be formulated for direct application or formulatedas a concentrated form of a primary composition which needs to bediluted prior to use. Optionally, the composition can be provided in theform of a kit comprising the interfering ribonucleic acid or a host cellcomprising/expressing the interfering ribonucleic acid in a container,and a suitable diluent or carrier for the RNA or host cell in a separatecontainer. In the application of the present invention, the compositioncan be applied to a plant or any part thereof in any development stageof the plant, for example, during the culture of the plant in a field,the composition is applied to the aboveground part of the plant; or whenthe plant seeds are stored or after the plant seeds are sown in thesoil, the composition is applied to the plant seeds. In general, it isimportant to achieve a good control over an insect in an early growthstage of the plant, since this stage is a period when the plant ispossibly suffering from most serious insect damage.

In the present invention, the composition can be applied to theenvironment of insects through different techniques which include, butare not limited to, spraying, atomizing, dusting, scattering, pouring,seed coating, seed treatment, introduction into the soil andintroduction into irrigation water. When a plant which is sensitive toinsect infestations is treated, the composition can be delivered to theplant or a part thereof before the occurrence of the insect (for apreventative purpose) or after the emergence of signs of an insectinvasion (for a control purpose).

The composition of the present invention can be formulated as comprisingat least one additional active agent. Therefore, the composition can beprovided in the form of a “multi-part kit”, and the kit comprises acomposition comprising an interfering ribonucleic acid in a container,and one or more suitable active components, such as chemical orbiological pesticides, in a separate container. Optionally, thecomposition can be provided in the form of a mixture which is stable andthe components of which can be used in combination with each other.

Suitable active components which can be used in a complementary mannerwith the interfering ribonucleic acid of the present invention include,but are not limited to, the following items: dursban, allethrin,resmethrin, tetrabromoethyl, dimethanol-cyclopropanecarboxylic acid(generally being comprised in a liquid composition); and hydramethylnon,avermectin, dursban, sulfluramid, hydroprene, fipronil (a GABAreceptor), carbamic acid isopropyl phenyl methyl ester, indoxacarb,noviflumuron (a chitin synthesis inhibitor), imiprothrin, abamectin (aglutamate gated chloride ion channel), and imidacloprid (anacetylcholine receptor) (generally being comprised in a baitcomposition). Preferably, taking the health and environment intoaccount, it is known that the active component is a pesticide such ashydramethylnon and avermectin.

The composition in the present invention can be formulated as comprisingat least one additional agronomical reagent, such as a herbicide or anadditional pesticide. The term “additional pesticide” or “a secondpesticide” refers to a pesticide apart from the first or initialinterfering RNA molecule of the composition. Optionally, the compositionof the present invention can be delivered in combination with at leastone additional agronomical reagent (for example a herbicide or a secondpesticide). The composition can be provided in combination with aherbicide which is selected from any herbicide known in the art, forexample, glyphosate, 2,4-D, imidazolinone, sulfonylurea and bromoxynil.The composition can also be provided in combination with at least oneadditional pesticide which can be selected from any pesticide known inthe art and/or can comprise an interfering ribonucleic acid whichfunctions to down-regulate the expression of a target gene in an insectafter being ingested by the insect. The target pest is an insect and theinterfering ribonucleic acid is selected from any one of the interferingribonucleic acids in the present invention. The additional pesticidecomprises an interfering ribonucleic acid which functions todown-regulate the expression of a known gene in any target pest. Theinitial interfering ribonucleic acid and the second or additionalpesticide in the composition can be targeted at the same or differentinsects. For example, the initial interfering ribonucleic acid and thesecond pesticide can be targeted at different insects or can be targetedat insects of different families or classes, for example fungi ornematodes or insects. A person skilled in the art should be clear on howto detect a synergistic effect of the combination of the interferingribonucleic acid and other agronomical reagents. Preferably, thecomposition comprises a first interfering ribonucleic acid and one ormore additional pesticides, each of which has a toxicity for the sameinsect, wherein the one or more additional pesticides are selected froma potato tuber-specific protein, a Bacillus thuringiensis insecticidalprotein, a Xenorhabdus ehlersii insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporus insecticidal protein, aBacillus sphaericus insecticidal protein and lignin. Differentcomponents can be delivered simultaneously or successively to a regionor organism to be treated.

The method for preventing and/or controlling insect invasions in thepresent invention comprises contacting an insect with an effectiveamount of at least one interfering ribonucleic acid, wherein theinterfering ribonucleic acid functions to down-regulate the expressionof a necessary target gene of insect after being ingested by the insect.The necessary target gene can be any gene of the insect involved in theregulating of the initiation or maintenance of necessary biologicalprocesses required for infestation in the insect, and the biologicalprocesses include, but are not limited to, survival, growth,development, reproduction and pathogenicity.

The method for preventing and/or controlling insect invasions in thecrop plant field in the present invention comprises expressing aneffective amount of the interfering ribonucleic acid in the plant, andin the case that the method is used for controlling insect invasions,the term “effective amount” refers to an amount or concentration of theinterfering ribonucleic acid required for producing a phenotypic effecton the insect, so that the number of the insects infesting a hostorganism is reduced and/or the amount of damage caused by the insect isdecreased. The phenotypic effect can be insect death, and the use of theinterfering RNA achieves an insect death rate of at least 20%, 30%, 40%,preferably at least 50%, 60%, 70%, and more preferably at least 80% or90% as compared with a control insect. The phenotypic effect can alsoinclude, but is not limited to, the prevention of insect growth, arrestof feeding activity and reduction of egg-laying. Therefore, as comparedwith the control insect, the total number of the insects invading thehost organism can be reduced by at least 20%, 30%, 40%, preferably by atleast 50%, 60%, 70%, and more preferably by at least 80% or 90%.Optionally, as compared with the control insect, the damage caused bythe insect can be reduced by at least 20%, 30%, 40%, preferably by atleast 50%, 60%, 70%, and more preferably by at least 80% or 90%.Therefore, the present invention can be used to achieve at least 20%,30%, 40%, preferably at least 50%, 60%, 70%, and more preferably atleast 80% or 90% of control of the insect.

The method and composition in the present invention can be used torestrict or eliminate the invasion of a Coleoptera pest, preferablyMonolepta hieroglyphica (Motschulsky), in the environment or on thesurface where any pest host, pest symbiont or pest may be present, byproviding one or more compositions comprising the dsRNA molecules in thepresent invention in the food of the pest. The method is especiallybeneficial for preventing the insect from attacking a plant, and thepest is defined as having a pH of about 4.5 to about 9.5, about 5 toabout 9, about 6 to about 7 or about pH 7.0 in the digestive system.

The nucleotide sequence of the present invention can comprise invertedrepeats spaced apart by a “spacer sequence”. The spacer sequence can bea region comprising any of the following nucleotide sequences, ifdesired, which can promote the formation of a secondary structurebetween each segment of repeats. The spacer sequence is a part for asense or antisense coding sequence of mRNA. Alternatively, the spacersequence can comprise any combination of nucleotides or homologuesthereof which can be covalently linked to a nucleic acid molecule. Thespacer sequence can comprise a nucleotide sequence with a length of atleast about 10-100 nucleotides, or a length of at least about 100-200nucleotides, or a length of at least about 200-400 nucleotides, or alength of at least about 400-500 nucleotides.

In the present invention, the “introduction” of the interferingribonucleic acid into a plant means introduction that can be performedby a direct transformation method, for example, Agrobacterium-mediatedtransformation for a plant tissue, microparticle bombardment,electroporation, etc.; or introduction that can be performed byhybridizing a plant having a heterogenous nucleotide sequence withanother plant, so that the progenies have the nucleotide sequenceincorporated into their genomes. Such breeding technologies are wellknown to a person skilled in the art.

The present invention provides a polynucleotide and method forcontrolling insect invasions, at least having the following advantages:

1. The present invention discloses, for the first time, a plurality oftarget sequences for controlling the target gene c4506 of an insect pestof Coleoptera, Monolepta hieroglyphica (Motschulsky), and furthermore,verifies that a nucleic acid inhibitor obtained based on these targetsequences can be directly used for controlling invasions of insect pestsof Coleoptera.2. High species specificity. The target sequences disclosed herein forcontrolling an insect pest of Coleoptera, Monolepta hieroglyphica(Motschulsky), act with high specificity on Monolepta hieroglyphica(Motschulsky) and species that share close genetic affinities and havesequence identity.3. Avoidance of development of resistance. The present invention doesnot rely on the binding of a specific dsRNA to a receptor protein in aninsect body, and thus can effectively avoid the analogous risk ofdeveloping resistance to Bt-toxin proteins in the insect.4. The RNAi technology used herein is highly efficient and specific, andthe dsRNA obtained can be directly used in field for controlling theinvasion of insect pests of Coleoptera, which is convenient, inexpensivein cost, and good in environment compatibility.

The technical solutions of the present invention are further describedin details through drawings and examples below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electrophoretogram showing the expression level of thetarget gene c4506 used in the polynucleotide and method for controllinginsect invasions according to the present invention.

FIG. 2 is a schematic diagram of recombinant expression vectorDBNR4506C1 used in the polynucleotide and method for controlling insectinvasions according to the present invention.

DETAILED DESCRIPTION

The technical solution of the polynucleotide and method for controllinginsect invasions in the present invention is further illustrated by thespecific examples below.

Example 1. Determination of Target Sequences of Monolepta hieroglyphica(Motschulsky)

1. Total RNA Extraction of Monolepta hieroglyphica (Motschulsky)

Newly-incubated instar larvae of Monolepta hieroglyphica (Motschulsky)were taken as materials, and RNA was extracted by using the conventionalTrizol method, purified by a conventional method, and treated with aDNase, thereby obtaining a total RNA sample at a concentration of ≥300ng/μL a total amount of ≥6 μg, and OD_(260/280) of 1.8-2.2.

2. Separation of mRNA and Synthesis of cDNA

mRNA with polyA was separated from the total RNA sample prepared asabove using magnetic beads with oligo-dT, and the first strand of cDNAwas then synthesized using a random hexamer and a Superscript II reversetranscriptase kit of Invitrogen.

3. Screening of Target Genes

One target gene c4506 of Monolepta hieroglyphica (Motschulsky) wasscreened out from the genes that are in the larvae library with mediumanalytical expression value and may be involved in important metabolicpathways, and its full-length nucleotide sequence was shown in SEQ IDNO: 1, the amino acid sequence was shown in SEQ ID NO: 2.

4. Selection of Target Sequences within the Target Genes

Four target sequences with different ORF positions and/or differentlengths of the target gene c4506 were selected, as shown in Table 1.

TABLE 1 sequence information of four target sequences Target sequenceSequence number c4506_g1-01 SEQ ID NO: 3 c4506_g1-02 SEQ ID NO: 4c4506_g1-03 SEQ ID NO: 5 c4506_g1-04 SEQ ID NO: 6

Example 2. Acquisition of dsRNA

The dsRNA of the above-mentioned four target sequences were synthesizedrespectively according to the instructions of MEGAscript RNAi Kit fromThermoFisher company, namely, c4506_g1-01 to c4506_g1-04; the size ofthe products were detected by agarose electrophoresis with a massconcentration of 1%, and the concentrations of c4506_g1-01 toc4506_g1-04 were determined respectively by NanoDrop 2000 (ThermoScientific).

Example 3. Identification of the Ability of Controlling Monoleptahieroglyphica (Motschulsky) by Feeding dsRNA

The isolated and purified c4506_g1-01 to c4506_g1-04 were mixedrespectively and added evenly into feed at the ratios of 50 μg/g feedand 5 μg/g feed (Feed formula references Development of an artificialdiet for the western corn rootworm, Entomologia Experimentalis etApplicata 105: 1-11, 2002.), to obtain c4506_g1-01-50 to c4506_g1-04-50feed and c4506_g1-01-5 to c4506_g1-04-5 feed, respectively. In thecontrol group, irrelevant dsRNA (SEQ ID NO: 15) was added to the feedCK, and other conditions were completely consistent. The newly-incubatedlarvae of Monolepta hieroglyphica (Motschulsky) were fed with the feedprepared as above. 30 newly-incubated larvae with an incubation time ofnot more than 24 hours were placed in each dish, in which the feed mixedwith dsRNA was replaced every two days and fed until day 14. The insectmortality rate was counted every two days from the beginning of feeding,and the expression value of the target gene was determined on days 0, 4,8, 10, 12 and 14 from the beginning of feeding, by using the specificmethods as follows:

Step 301. The larvae, fed with c4506_g1-01-50 to c4506_g1-04-50 feed andc4506_g1-01-5 to c4506_g1-04-5 feed respectively, were collected on days0, 4, 8, 10, 12 and 14, respectively, and frozen with liquid nitrogen;

Step 302. The total RNA of the above-mentioned larvae was extractedusing the Trizol method, respectively;

Step 303. The cDNA was obtained by reverse transcription of the totalRNA of the above-mentioned larvae using the whole gold kit (TransGenBiotech ER301-01), respectively.

Step 304. Ubiquitin-C was used as an internal reference gene for PCRamplification, and after amplification, 10 μL of the amplificationproduct was taken for agarose gel electrophoresis with a massconcentration of 1%.

Five repeats were set for each treatment in the above-mentionedexperiment, and the statistical results were shown in FIG. 1 and Table2. In Table 2, “−50” in the material number represents 50 μg of thecorresponding dsRNA per g of feed, i.e., “50 μg/g feed” as previouslystated; “−5” represents 5 μg of the corresponding dsRNA per gram offeed, i.e., “5 μg/g feed” as previously stated. For example,“r1-dsRNA-50” represents 50 μg r1-dsRNA per gram of feed. “DAI”represents the number of days after incubating and feeding the insects.

The measured results of expression amount of the target gene in FIG. 1showed that dsRNA (50 μg/g feed) of the target sequence c4506_g1-01 hadsignificant inhibition effect on the expression of the target gene c4506in the Monolepta hieroglyphica (Motschulsky), and the expression amountof the target gene c4506 was significantly down-regulated on day 10 offeeding, the expression of the target gene c4506 were almost notdetected on day 14.

The results of feeding with dsRNA in Table 2 showed that the dsRNA oftarget sequences c4506_g1-01 to c4506_g1-04 of the target gene c4506 hadsignificant lethal effect on the Monolepta hieroglyphica (Motschulsky),and there were no surviving larvae in most repeats on day 14 of feeding.

TABLE 2 Experimental results of survival rate of Monolepta hieroglyphica(Motschulsky) fed with dsRNA Material Number DAI0 DAI2 DAI4 DAI6 DAI8DAI10 DAI12 DAI14 CK-dsRNA 100% ± 0% 100% ± 0% 98% ± 3% 95% ± 4% 91% ±8% 88% ± 9% 85% ± 11% 83% ± 11% c4506_g1-01-50 100% ± 0% 100% ± 0% 98% ±3% 96% ± 4% 86% ± 9% 46% ± 8% 31% ± 10% 12% ± 12% c4506_g1-01-5 100% ±0% 100% ± 0% 98% ± 1% 95% ± 3%  88% ± 10%  65% ± 10% 45% ± 8%  32% ± 15%c4506_g1-02-50 100% ± 0% 100% ± 0% 99% ± 2% 98% ± 3% 91% ± 7%  70% ± 12%53% ± 10% 39% ± 12% c4506_g1-02-5 100% ± 0% 100% ± 0% 100% ± 0%  98% ±2% 92% ± 8% 87% ± 9% 73% ± 7%  59% ± 12% c4506_g1-03-50 100% ± 0% 100% ±0% 99% ± 2% 96% ± 3% 88% ± 6% 50% ± 8% 42% ± 12% 21% ± 11% c4506_g1-03-5100% ± 0% 100% ± 0% 99% ± 1% 95% ± 4% 91% ± 6% 68% ± 8% 48% ± 11% 29% ±12% c4506_g1-04-50 100% ± 0% 100% ± 0% 98% ± 3% 97% ± 4% 90% ± 7% 66% ±9% 44% ± 10% 18% ± 12% c4506_g1-04-5 100% ± 0% 100% ± 0% 98% ± 2% 95% ±5% 92% ± 5% 73% ± 8% 49% ± 9%  36% ± 10%

Example 4. Unexpected Technical Effect of Interfering with the Same GeneExpression in Different Insects

Signal recognition particle 54 kDa protein, which belongs to one of thepeptide chains in the signal recognition particle complex, and its mainfunction is that when the pre-secreted protein is exposed from theribosome, signal recognition particle 54 kDa protein rapidly binds tothe signal sequence of the pre-secreted protein and transfers it to thetranslocation chain related membrane protein. The related literatureshowed that interfering with coding gene expression of signalrecognition particle 54 kDa protein can have lethal effects on a varietyof Coleoptera insects, as reported by Julia Ulrich et al. (2015), RNAiinterference was performed on the coding gene of the protein in theTribolium castaneum by an injection manner (injection sequence code ofiB_00404), and it was found that almost all Tribolium castaneum werekilled at about four days after injection. As also reported byAvet-Rochex et al. (2010), RNAi interference was performed on the codinggene of the protein in Drosophila by an injection manner (Table 1), andthe results showed that almost all Drosophila were killed afterinjection.

On the basis of the reports in the above-mentioned literatures and thehigh homology of the sequences, the coding gene of this protein inMonolepta hieroglyphica (Motschulsky) was screened out. As for sequencesfor injection into Tribolium castaneum and Drosophila, the sequence M1at corresponding position was selected, as shown in SEQ ID NO: 16, andthe sequence M2 at non-corresponding position was selected, as shown inSEQ ID NO: 17. The control ability for Monolepta hieroglyphica(Motschulsky) was determined by using a method of feeding dsRNA (at aratio of 50 μg/g of feed) in the Example 3 of the present invention. Asshown in Table 3, the experimental results showed that neither thesequence M1 at the corresponding position, nor the sequence M2 at thenon-corresponding position can produce a significant lethal effect onMonolepta hieroglyphica (Motschulsky), which was basically no differentfrom the control group. Similar experimental results were confirmed inPCT international public patent WO 2018/026770, which was verified withRNAi lethal genes of nematodes, Drosophila and so on after transcriptomewas obtained, that is, according to the known several lethal genes ofnematodes and Drosophila, RNAi interference was performed on thecorresponding gene in maize rootworm, and there was basically nosignificant lethal effect. In summary, the technical effect ofinterfering with the same gene expression of different insects wasunpredictable, and it is not inevitably associated with the technicaleffect of known interference and the homology of sequences.

TABLE 3 Experimental results of lethality rate of Monoleptahieroglyphica (Motschulsky) fed with dsRNA Material Number DAI4 DAI6DAI8 DAI10 DAI12 DAI14 CK-dsRNA 96% ± 6% 85% ± 9% 75% ± 16% 71% ± 16%69% ± 13% 69% ± 14% M1-dsRNA-50 98% ± 3% 92% ± 6% 89% ± 7%  83% ± 9% 69% ± 15% 63% ± 18% M2-dsRNA-50 91% ± 8%  88% ± 10% 84% ± 11% 76% ± 13%69% ± 15% 67% ± 17%

Example 5. Construction of Plant Expression Vectors

Two expression cassettes were synthesized according to the order ofp35S-RX-tNos-p35S-Hpt-tNos (X is 1-4), and connected to the plantexpression vectors through EcoR V and BamH I, and named DBNR4506CX (X is1-4, in which the vector schematic diagram of DBNR4506C1 was shown inFIG. 2 (Kan: Kanamycin gene; RB: the right boundary; pr35S: cauliflowermosaic virus 35S (SEQ ID NO: 7); R1 (SEQ ID NO: 8): the g1_01 nucleotidesequence (g1_01 is the target sequence 1 of target gene c4506, SEQ IDNO: 3)+spacer sequence (SEQ ID NO: 9)+the reverse complementary sequenceof the g1_01 nucleotide sequence; tNos: the terminator of nopalinesynthase gene (SEQ ID NO: 10); Hpt: hygromycin phosphotransferase gene(SEQ ID NO: 11); and LB: the left border).

Escherichia coli T1 competent cells were transformed with therecombinant expression vector DBNR4506C1 by a heat shock method with thefollowing heat shock conditions: water bathing 50 μL of Escherichia coliT1 competent cells and 10 μL of plasmid DNA (recombinant expressionvector DBNR4506C1) at 42° C. for 30 s; shake culturing at 37° C. for 1 h(using a shaker at a rotation speed of 100 rpm for shaking); thenculturing under the condition of a temperature of 37° C. for 12 h on aLB solid plate (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L ofNaCl, and 15 g/L of agar, adjusted to a pH of 7.5 with NaOH) containing50 mg/L of Kanamycin, picking white colonies, and culturing under theconditions of a temperature of 37° C. overnight in a LB liquid culturemedium (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl, and50 mg/L of Kanamycin, adjusted to a pH of 7.5 with NaOH). The plasmidsin the cells were extracted through an alkaline method: centrifuging thebacteria solution at a rotation speed of 12000 rpm for 1 min, removingthe supernatant, and the precipitated bacteria were suspended with 100μL of an ice precooled solution I (25 mM of Tris-HCl, 10 mM of EDTA(ethylenediamine tetraacetic acid), 50 mM of glucose, pH 8.0); adding200 μL of a freshly prepared solution II (0.2 M of NaOH, 1% SDS (sodiumdodecyl sulfate)), reversing the tube 4 times, mixing, and placing onice for 3-5 min; adding 150 μL of a cold solution III (3M of potassiumacetate, 5M of acetic acid), mixing evenly well immediately, and placingon ice for 5-10 min; centrifuging under the conditions of a temperatureof 4° C. and a rotation speed of 12000 rpm for 5 min, adding 2 times ofvolume of anhydrous ethanol to the supernatant, mixing evenly andplacing at room temperature for 5 min; centrifuging under the conditionsof a temperature of 4° C. and a rotation speed of 12000 rpm for 5 min,discarding the supernatant, and washing the precipitate with ethanol ata concentration (V/V) of 70% and drying; adding 30 μL of TE (10 mM ofTris-HCl, 1 mM of EDTA, pH 8.0) containing RNase (20 μg/mL) to dissolvethe precipitate; water bathing at 37° C. for 30 min to digest RNA;storing at −20° C. for later use. The extracted plasmids were sequencedand identified through PCR, and the results demonstrated that therecombinant expression vector DBNR4506C1 was correctly constructed.

According to the above-mentioned method, recombinant expression vectorsDBNR4506C2-DBNR4506C4 were constructed respectively, with the followingvector structures: Kan: Kanamycin gene; RB: the right boundary; pr35S:cauliflower mosaic virus 35S (SEQ ID NO: 7); RX: the g1_0X nucleotidesequence (g1_0X is the target sequence X of target gene c4506, X is2-4)+spacer sequence (SEQ ID NO: 9)+the reverse complementary sequenceof the g1_0X nucleotide sequence); tNos: the terminator of nopalinesynthase gene (SEQ ID NO: 10); Hpt: hygromycin phosphotransferase gene(SEQ ID NO: 11); and LB: the left boundary. Escherichia coli T1competent cells were transformed respectively with the recombinantexpression vector DBNR4506C2-DBNR4506C4 by a heat shock method, and theplasmids in the cells were extracted through an alkaline method.

Example 6. Transformation of Agrobacterium with the RecombinantExpression Vectors

Agrobacterium LBA4404 (Invitrogen, Chicago, USA, CAT: 18313-015) wastransformed respectively with the recombinant expression vectorsDBNR4506C1-DBNR4506C4 which had been correctly constructed, by using aliquid nitrogen method with the following transformation conditions:placing 100 μL of Agrobacterium LBA4404, and 3 μL of plasmid DNA(recombinant expression vector) in liquid nitrogen for 10 min, and warmwater bathing at 37° C. for 10 min; inoculating the transformedAgrobacterium LBA4404 into a LB tube, culturing under the conditions ofa temperature of 28° C. and a rotation speed of 200 rpm for 2 h, andthen spreading on a LB plate containing 50 mg/L of rifampicin and 100mg/L of Kanamycin until positive single clones were grown, picking outsingle clones for culturing and extracting the plasmids thereof, andperforming verification by enzyme digestion on the recombinantexpression vectors DBNR4506C1-DBNR4506C4 with restriction endonucleasesEcoR V and BamH I, with the results demonstrating that the structures ofthe recombinant expression vectors DBNR4506C1-DBNR4506C4 were completelycorrect.

Example 7. Acquisition of Transgenic Maize Plants

According to the conventionally used Agrobacterium infection method,young embryos of maize variety Zong31 (Z31) cultured under sterileconditions were co-cultured with the transformed Agrobacterium inExample 6, so as to transfer T-DNA (comprising the RX nucleotidesequence, a promoter sequence of a cauliflower mosaic virus 35S gene, aHpt gene and a Nos terminator sequence) in the recombinant expressionvectors DBNR4506C1-DBNR4506C4 constructed in Example 5 into the maizechromosome, thereby obtaining maize plants with the RX nucleotidesequence (X is 1-4) incorporated; meanwhile, wild type maize plants wereused as the control.

As regards the Agrobacterium-mediated maize transformation, briefly,immature young embryos were separated from maize, and contacted with anAgrobacterium suspension, wherein the Agrobacterium can transfer the RXnucleotide sequence to at least one cell of one of the young embryos(step 1: the infection step). In this step, the young embryos werepreferably immersed in an Agrobacterium suspension (OD₆₆₀=0.4-0.6, aculture medium for infection (4.3 g/L of MS salt, MS vitamin, 300 mg/Lof casein, 68.5 g/L of sucrose, 36 g/L of glucose, 40 mg/L ofacetosyringone (AS), and 1 mg/L of 2,4-dichlorophenoxyacetic acid(2,4-D), pH 5.3)) to initiate the inoculation. The young embryos wereco-cultured with Agrobacterium for a period of time (3 days) (step 2:the co-culturing step). Preferably, the young embryos were cultured in asolid culture medium (4.3 g/L of MS salt, MS vitamin, 300 mg/L ofcasein, 20 g/L of sucrose, 10 g/L of glucose, 100 mg/L of acetosyringone(AS), 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), and 8 g/L ofagar, pH 5.8) after the infection step. After this co-culturing stage,there can be an optional “recovery” step. In the “recovery” step, theremay be at least one antibiotic (cephalosporin) known to inhibit thegrowth of Agrobacterium in a culture medium for recovery (4.3 g/L of MSsalt, MS vitamin, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/L of2,4-dichlorophenoxyacetic acid (2,4-D), and 3 g/L of phytagel, pH 5.8),without the addition of a selective agent for plant transformant (step3: the recovery step). Preferably, the young embryos were cultured in asolid culture medium with the antibiotic, but without the selectiveagent, to eliminate Agrobacterium and provide a recovery stage for theinfected cells. Subsequently, the inoculated young embryos were culturedin a culture medium containing a selective agent (hygromycin), andgrowing transformed calli were selected (step 4: the selection step).Preferably, the young embryos were cultured in a solid culture mediumfor screening (4.3 g/L of MS salt, MS vitamin, 300 mg/L of casein, 30g/L of sucrose, 50 mg/L of hygromycin, 1 mg/L of2,4-dichlorophenoxyacetic acid (2,4-D), and 3 g/L of phytagel, pH 5.8)with the selective agent, resulting in selective growth of transformedcells. Then, plants were regenerated from the calli (step 5: theregeneration step). Preferably, the calli grown on a culture mediumcontaining the selective agent were cultured in solid culture media (MSdifferentiation culture medium and MS rooting culture medium) toregenerate plants.

The resistant calli obtained from screening were transferred onto the MSdifferentiation culture medium (4.3 g/L of MS salt, MS vitamin, 300 mg/Lof casein, 30 g/L of sucrose, 2 mg/L of 6-benzyladenine, 50 mg/L ofhygromycin, and 3 g/L of phytagel, pH 5.8), and cultured at 25° C. fordifferentiation. The differentiated seedlings were transferred onto theMS rooting culture medium (2.15 g/L of MS salt, MS vitamin, 300 mg/L ofcasein, 30 g/L of sucrose, 1 mg/L of indole-3-acetic acid, and 3 g/L ofphytagel, pH 5.8), cultured at 25° C. until reaching a height of about10 cm, and transferred to a greenhouse for culturing until fruiting. Inthe greenhouse, the plants were cultured at 28° C. for 16 hours, andthen cultured at 20° C. for 8 hours every day.

Example 8. Acquisition of Transgenic Soybean Plants

According to the conventionally used Agrobacterium infection method,cotyledonary node tissues of soybean variety Zhonghuang13 cultured understerile conditions were co-cultured with the transformed Agrobacteriumin Example 6, so as to transfer T-DNA (comprising the RX nucleotidesequence, a promoter sequence of a cauliflower mosaic virus 35S gene, aHpt gene and a Nos terminator sequence) in the recombinant expressionvectors DBNR4506C1-DBNR4506C4 constructed in Example 5 into the soybeanchromosome, thereby obtaining soybean plants with the RX nucleotidesequence (X is 1-4) incorporated; meanwhile, wild type soybean plantswere used as the control.

As regards the Agrobacterium-mediated soybean transformation, briefly,mature soybean seeds were germinated in a culture medium for soybeangermination (3.1 g/L of B5 salt, B5 vitamin, 20 g/L of sucrose, and 8g/L of agar, pH 5.6), and the seeds were inoculated on the culturemedium for germination and cultured under the conditions of atemperature of 25±1° C.; and a photoperiod (light/dark) of 16 h/8 h.After 4-6 days of germination, soybean sterile seedlings swelling atbright green cotyledonary nodes were taken, hypocotyls were cut off 3-4mm below the cotyledonary nodes, the cotyledons were cut longitudinally,and apical buds, lateral buds and seminal roots were removed. A woundwas made at a cotyledonary node using the knife back of a scalpel, thewounded cotyledonary node tissues were contacted with an Agrobacteriumsuspension, wherein the Agrobacterium can transfer the RX nucleotidesequence to the wounded cotyledonary node tissues (step 1: the infectionstep). In this step, the cotyledonary node tissues were preferablyimmersed in the Agrobacterium suspension (OD₆₆₀=0.5-0.8, a culturemedium for infection (2.15 g/L of MS salt, B5 vitamin, 20 g/L ofsucrose, 10 g/L of glucose, 40 mg/L of acetosyringone (AS), 4 g/L of2-morpholine ethanesulfonic acid (MES), and 2 mg/L of zeatin (ZT), pH5.3)) to initiate the inoculation. The cotyledonary node tissues wereco-cultured with the Agrobacterium for a period of time (3 days) (step2: the co-culturing step). Preferably, the cotyledonary node tissueswere cultured in a solid culture medium (4.3 g/L of MS salt, B5 vitamin,20 g/L of sucrose, 10 g/L of glucose, 4 g/L of 2-morpholineethanesulfonic acid (MES), 2 mg/L of zeatin, and 8 g/L of agar, pH 5.6)after the infection step. After this co-culturing stage, there can be anoptional “recovery” step. In the “recovery” step, there may be at leastone antibiotic (cephalosporin) known to inhibit the growth ofAgrobacterium in a culture medium for recovery (3.1 g/L of B5 salt, B5vitamin, 1 g/L of 2-morpholine ethanesulfonic acid (MES), 30 g/L ofsucrose, 2 mg/L of zeatin (ZT), 8 g/L of agar, 150 mg/L ofcephalosporin, 100 mg/L of glutamic acid, and 100 mg/L of aspartic acid,pH 5.6), without the addition of a selective agent for planttransformant (step 3: the recovery step). Preferably, tissue blocksregenerated from the cotyledonary nodes were cultured in a solid culturemedium with the antibiotic, but without the selective agent, toeliminate Agrobacterium and provide a recovery stage for the infectedcells. Subsequently, the tissue blocks regenerated from the cotyledonarynodes were cultured in a culture medium containing a selective agent(hygromycin), and growing transformed calli were selected (step 4: theselection step). Preferably, the tissue blocks regenerated from thecotyledonary nodes were cultured in a solid culture medium for screening(3.1 g/L of B5 salt, B5 vitamin, 1 g/L of 2-morpholine ethanesulfonicacid (MES), 30 g/L of sucrose, 1 mg/L of 6-benzyladenine (6-BAP), 8 g/Lof agar, 150 mg/L of cephalosporin, 100 mg/L of glutamic acid, 100 mg/Lof aspartic acid, and 50 mg/L of hygromycin, pH 5.6) with the selectiveagent, resulting in selective growth of transformed cells. Then, plantswere regenerated from the transformed cells (step 5: the regenerationstep). Preferably, the tissue blocks regenerated from the cotyledonarynodes grown on a culture medium containing the selective agent werecultured in solid culture media (B5 differentiation culture medium andB5 rooting culture medium) to regenerate plants.

The resistant tissue blocks obtained from screening were transferredonto the B5 differentiation culture medium (3.1 g/L of B5 salt, B5vitamin, 1 g/L of 2-morpholine ethanesulfonic acid (MES), 30 g/L ofsucrose, 1 mg/L of zeatin (ZT), 8 g/L of agar, 150 mg/L ofcephalosporin, 50 mg/L of glutamic acid, 50 mg/L of aspartic acid, 1mg/L of gibberellin, 1 mg/L of auxin, and 50 mg/L of hygromycin, pH5.6), and cultured at 25° C. for differentiation. The differentiatedseedlings were transferred onto the B5 rooting culture medium (3.1 g/Lof B5 salt, B5 vitamin, 1 g/L of 2-morpholine ethanesulfonic acid (MES),30 g/L of sucrose, 8 g/L of agar, 150 mg/L of cephalosporin, and 1 mg/Lof indole-3-butyric acid (IBA)), cultured on the rooting culture mediumat 25° C. until reaching a height of about 10 cm, and transferred to agreenhouse for culturing until fruiting. In the greenhouse, the plantswere cultured at 26° C. for 16 hours, and then cultured at 20° C. for 8hours every day.

Example 9. Verification of the Transgenic Maize Plants and theTransgenic Soybean Plants Using TaqMan

About 100 mg of leaves from the maize plants into which the RXnucleotide sequence (X is 1-4) was incorporated, were taken as samples.The genomic DNA thereof was extracted with a DNeasy Plant Maxi Kit fromQiagen respectively, and the copy number of a Hpt gene was detected bythe Taqman probe fluorescence quantitative PCR method so as to determinethe copy numbers of the RX nucleotide sequence. Meanwhile, wild typemaize plants were used as the control, and detected and analyzedaccording to the above-mentioned method. Triple repeats were set for theexperiments, and were averaged.

The particular method for detecting the copy number of the Hpt gene wasas follows:

Step 901. 100 mg of leaves from the maize plants into which the RXnucleotide sequence was incorporated and wild type maize plants wererespectively taken, ground into a homogenate in a mortar with liquidnitrogen, and triple repeats were taken for each sample;

Step 902. The genomic DNA of the above-mentioned samples was extractedusing a DNeasy Plant Mini Kit from Qiagen, and the particular method canrefer to the product instruction thereof;

Step 903. The concentrations of the genomic DNAs of the above-mentionedsamples were detected using NanoDrop 2000 (Thermo Scientific);

Step 904. The concentrations of the genomic DNAs of the above-mentionedsamples were adjusted to a consistent concentration value which rangesfrom 80-100 ng/μL;

Step 905. The copy numbers of the samples were identified using theTaqman probe fluorescence quantitative PCR method, wherein samples forwhich the copy numbers had been identified and known were taken asstandards, the samples of the wild type maize plants were taken as thecontrol, and triple repeats were taken for each sample, and wereaveraged; the sequences of the primers and probe for fluorescencequantitative PCR were as follows, respectively:

The following primers and probe were used for detecting the Hptnucleotide sequence:

Primer 1: cagggtgtcacgttgcaaga as shown in SEQ ID NO: 12 of the sequencelisting;

Primer 2: ccgctcgtctggctaagatc as shown in SEQ ID NO: 13 of the sequencelisting;

Probe 1: tgcctgaaaccgaactgcccgctg as shown in SEQ ID NO: 14 of thesequence listing;

PCR Reaction System:

JumpStart ™ Taq ReadyMix ™ (Sigma) 10 μL 50× primer/probe mixture  1 μLgenomic DNA  3 μL water (ddH₂O)  6 μL

The 50×primer/probe mixture comprises 45 μL of each primer at aconcentration of 1 mM, 50 μL of the probe at a concentration of 100 μM,and 860 μL of 1×TE buffer, and was stored at 4° C. in an centrifugetube.

PCR Reaction Conditions:

Step Temperature Time 911 95° C.  5 min 912 95° C. 30 s 913 60° C.  1min 914 back to step 912, repeated 40 times

Data was analyzed using software SDS2. 3 (Applied Biosystems).

By analyzing the experimental results of the copy number of the Hptgene, it was further demonstrated whether the RX nucleotide sequence wasrespectively incorporated into the chromosome of the detected maizeplants, and whether the maize plants into which the RX nucleotidesequence (X is 1-4) was incorporated resulted in single-copy transgenicmaize plants.

According to the above-mentioned method of verifying the transgenicmaize plants using TaqMan, the transgenic soybean plants were detectedand analyzed. It was further demonstrated, by analyzing the experimentalresults of the copy number of the Hpt gene, that the RX nucleotidesequence was incorporated into the chromosomes of the detected soybeanplants, and the soybean plants into which the RX nucleotide sequence (Xis 1-4) was incorporated resulted in single-copy transgenic soybeanplants.

Example 10. Identification of Insecticidal Effect of Transgenic Maize onMonolepta hieroglyphica (Motschulsky)

The insecticidal effect against Monolepta hieroglyphica (Motschulsky) ofthe maize plants into which the RX nucleotide sequence (X is 1-4) wasincorporated was detected.

Step 1001. Ten strains of DBNR4506C1-DBNR4506C4 maize transformationevents (RX-M), each of which was identified as a positive single copythrough taqman, and three strains of maize transformation events (NGM1)which were identified as negative through taqman were chosen; meanwhile,wild type maize plants were used as the control (CK1); and the plantswere grown in a greenhouse until trefoil stage;

Step 1002. The materials in step 1001 were taken, and a third young leafwas taken from each seedling, and cut to a size of 1×2 cm of leaf inwhich the main vein was removed, and laid and placed in a culture dishwith a moist filter paper laid thereon;

Step 1003. 10 newly-incubated larvae of Monolepta hieroglyphica(Motschulsky) with an incubation time of not more than 24 h were placedin each dish, the covers of the dishes covered same tightly, the culturedishes were placed in a bioassay box with a moist piece of gauze laid atthe bottom thereof, and the bioassay box was placed in a bioassaychamber at a temperature of 24±2° C., D/L of 24/0, and a humidity of70%-80%;

Step 1004. Considering that the newly-incubated larvae of Monoleptahieroglyphica (Motschulsky) are small and weak, and easily suffer frommechanical injuries, it was better to keep the culture dishes unmoved onthe day that the insects were incubated and 1 day after incubation;

Step 1005. Starting on day 2 after the incubation of the insects, thenumber of surviving Monolepta hieroglyphica (Motschulsky) was countedfrom the exterior of the culture dishes every day until the end of day16; insects of Monolepta hieroglyphica (Motschulsky) surviving in theculture dishes were transferred to culture dishes charged with freshleaves every two days, and the experimental results were shown in Table4.

TABLE 4 Experimental results of feeding Monolepta hieroglyphica(Motschulsky) with leaves having maize transformation events MaterialSurvival rate of Monolepta hieroglyphica (Motschulsky) at each two daysafter bioassay number DAI2 DAI4 DAI6 DAI8 DAI10 DAI12 DAI14 DAI16 CK1100% ± 0% 98% ± 4% 92% ± 4% 85% ± 8% 82% ± 9%  80% ± 8% 76% ± 9%  71% ±8%  NGM1 100% ± 0% 95% ± 2% 93% ± 5% 87% ± 7% 84% ± 10% 80% ± 8% 75% ±10% 72% ± 9%  R1-M 100% ± 0% 94% ± 5% 91% ± 4% 90% ± 6% 78% ± 10% 68% ±8% 56% ± 15% 50% ± 15% R2-M 100% ± 0% 94% ± 3% 92% ± 5% 87% ± 6% 80% ±5%  71% ± 7% 52% ± 12% 49% ± 11% R3-M 100% ± 0% 93% ± 5% 90% ± 3% 85% ±7% 78% ± 8%  69% ± 8% 50% ± 10% 48% ± 13% R4-M 100% ± 0% 94% ± 2% 88% ±2% 83% ± 6% 76% ± 5%  67% ± 9% 52% ± 9%  44% ± 9% 

The experimental results in Table 4 demonstrated that the maize plantsinto which the RX nucleotide sequence (X is 1-4) was incorporated hadgood inhibitory effects on Monolepta hieroglyphica (Motschulsky), andthe survival rate (survival rate=survival number/test number) ofMonolepta hieroglyphica (Motschulsky) was about 50% on day 16.

Example 11. Identification of Insecticidal Effect of Transgenic Soybeanon Monolepta hieroglyphica (Motschulsky)

The insecticidal effect against Monolepta hieroglyphica of the soybeanplants into which the RX nucleotide sequence (X is 1-4) was incorporatedwas detected.

Step 1101. Ten strains of DBNR4506C1-DBNR4506C4 soybean transformationevents (RX-S) each of which was identified as a positive single copythrough taqman, and three strains of soybean transformation events(NGM2) which were identified as negative through taqman were chosen;meanwhile, wild type soybean plants were used as the control (CK2); andthe plants were grown in a greenhouse until three pieces of euphyllawere grown;

Step 1102. The materials in step 1101 were taken, and a piece ofeuphylla with a size of about 2×2 cm was taken from each seedling, andlaid and placed in a culture dish with a moist filter paper laidthereon;

Step 1103. 15 newly-incubated larvae of Monolepta hieroglyphica(Motschulsky) with an incubation time of not more than 24 h were placedin each dish, the covers of the dishes covered same tightly, the culturedishes were placed in a bioassay box with a moist piece of gauze laid atthe bottom thereof, and the bioassay box was placed in a bioassaychamber at a temperature of 24±2° C., D/L of 24/0, and a humidity of70%-80%;

Step 1104. Considering that the newly-incubated larvae of Monoleptahieroglyphica (Motschulsky) are small and weak, and easily suffer frommechanical injuries, it was better to keep the culture dishes unmoved onthe day that the insects were incubated and 1 day after incubation;

Step 1105. Starting on day 2 after the incubation of the insects, thenumber of surviving Monolepta hieroglyphica (Motschulsky) was countedfrom the exterior of the culture dishes every day until the end of day16; insects of Monolepta hieroglyphica (Motschulsky) surviving in theculture dishes were transferred to culture dishes charged with fresheuphylla every two days, and the experimental results were shown inTable 5.

TABLE 5 Experimental results of feeding Monolepta hieroglyphica(Motschulsky) with euphylla having soybean transformation eventsMaterial Survival rate of Monolepta hieroglyphica (Motschulsky) at eachtwo days after bioassay number DAI2 DAI4 DAI6 DAI8 DAI10 DAI12 DAI14DAI16 CK2 100% ± 0% 100% ± 0% 95% ± 3% 94% ± 4%  90% ± 4%  86% ± 8%  80%± 9%  74% ± 8%  NGM2 100% ± 0% 100% ± 0% 96% ± 2% 95% ± 4%  92% ± 5% 88% ± 10% 78% ± 11% 72% ± 11% R1-S 100% ± 0%  95% ± 1% 92% ± 6% 91% ±10% 92% ± 8%  83% ± 15% 60% ± 9%  46% ± 9%  R2-S 100% ± 0%  98% ± 2% 93%± 4% 92% ± 8%  83% ± 12% 72% ± 9%  65% ± 12% 54% ± 12% R3-S 100% ± 0% 90% ± 4% 94% ± 7% 90% ± 11% 90% ± 12% 84% ± 14% 71% ± 13% 54% ± 13%R4-S 100% ± 0%  99% ± 0% 91% ± 7% 91% ± 11% 86% ± 11% 79% ± 12% 62% ±9%  58% ± 9% 

The experimental results in Table 5 demonstrated that the soybean plantsinto which the RX nucleotide sequence (X is 1-4) was incorporated hadgood inhibitory effects on Monolepta hieroglyphica (Motschulsky), andthe survival rate (survival rate=survival number/test number) ofMonolepta hieroglyphica (Motschulsky) was up to 58% on day 16.

Example 12. Composition

Formula of an agriculturally acceptable carrier for dsRNA (1 L system):50 mM of NaHPO₄ (pH7.0), 10 mM of β-mercaptoethanol, 10 mM of EDTA,sodium hexadecylsulfonate at a mass fraction of 0.1%, and polyethyleneglycol octyl phenyl ether at a mass fraction of 0.1%, make up to 1 Lwith H₂O.

The above-mentioned formula was a buffer formula, provided that anypurified dsRNA is directly added to the buffer so that the finalconcentration met requirements, such as 50 mg/L. The formula can also beprepared into a concentrated preparation as desired.

In summary, the present invention discloses, for the first time, atarget gene c4506 and target sequence thereof for controlling an insectpest of Coleoptera, Monolepta hieroglyphica (Motschulsky), andtransgenic plants (maize and soybean) obtained by using RNAi technology.The transgenic plants control the invasion of Monolepta hieroglyphica(Motschulsky) efficiently and specifically by introducing dsRNAsequences formed from the target sequences, and Monolepta hieroglyphica(Motschulsky) can be prevented from developing a similar risk ofBt-toxin protein resistance, with the advantages of good environmentcompatibility, convenience and low cost.

Finally, it should be stated that the above examples are merely used forillustrating, rather than limiting, the technical solution of thepresent invention; and although the present invention has beenillustrated in detail with reference to the preferred examples, a personskilled in the art should understand that modifications or equivalentreplacements may be made to the technical solution of the presentinvention without departing from the spirit and scope of the technicalsolution of the present invention.

What is claimed is:
 1. An isolated polynucleotide comprising aheterologous promoter operably linked to a polynucleotide, wherein thepolynucleotide comprises a polynucleotide sequence that is at least 99%identical to a fragment of SEQ ID NO:1 that is at least 121 nucleotidesin length, wherein when a Coleopteran insect pest ingests adouble-stranded RNA comprising at least one strand complementary to thepolynucleotide sequence, the growth of the Coleopteran insect pest isinhibited, and wherein the Coleopteran insect pest is Monoleptahieroglyphica.
 2. The polynucleotide according to claim 1, wherein thepolynucleotide also comprises a spacer sequence.
 3. The polynucleotideaccording to claim 2, wherein the spacer sequence is SEQ ID NO:
 9. 4. Anexpression cassette or a recombinant vector, comprising thepolynucleotide according to claim
 1. 5. An interfering ribonucleic acid,wherein the interfering ribonucleic acid acts to down-regulateexpression of at least one target gene in an insect pest of Monoleptahieroglyphica after being ingested by the insect pest, wherein theinterfering ribonucleic acid comprises at least one silencing element,wherein the silencing element is a double-stranded RNA region comprisingcomplementary strands which have been annealed, and one strand of whichcomprises a nucleotide sequence at least partially complementary to atarget sequence within the target gene, and the target sequencecomprises a polynucleotide sequence that is at least 99% identical to afragment of SEQ ID No:1 that is at least 121 nucleotides in length. 6.The interfering ribonucleic acid according to claim 5, wherein thesilencing element comprises a sequence of at least 15, 17, 19 or 21consecutive nucleotides complementary to or at least partiallycomplementary to a target sequence within the target gene.
 7. Theinterfering ribonucleic acid according to claim 5, wherein theinterfering ribonucleic acid comprises at least two silencing elements,each of which comprises a nucleotide sequence at least partiallycomplementary to a target sequence within the target gene.
 8. Theinterfering ribonucleic acid according to claim 7, wherein each of thesilencing elements comprises a different nucleotide sequencecomplementary to a different target sequence.
 9. The interferingribonucleic acid according to claim 8, wherein the different targetsequence is derived from a single target gene or from a target genedifferent from the target gene.
 10. The interfering ribonucleic acidaccording to claim 9, wherein the target gene different from the targetgene is derived from a same insect pest of Monolepta hieroglyphica or adifferent insect pest of Coleoptera.
 11. The interfering ribonucleicacid according to claim 5, wherein the interfering ribonucleic acid alsocomprises a spacer sequence.
 12. The interfering ribonucleic acidaccording to claim 11, wherein the spacer sequence is SEQ ID NO:
 9. 13.A composition for controlling invasion of an insect pest of Monoleptahieroglyphica, comprising at least one of the interfering ribonucleicacids according to claim 5 or a host cell expressing or capable ofexpressing the interfering ribonucleic acid sequence, and at least onesuitable carrier, excipient or diluent.
 14. The composition according toclaim 13, wherein the host cell is a bacterial cell.
 15. The compositionaccording to claim 13, wherein the composition is a solid, a liquid or agel.
 16. The composition according to claim 15, wherein the compositionis an insecticidal spray.
 17. The composition according to claim 13,wherein the composition also comprises at least one pesticide, whereinthe pesticide is a chemical pesticide, a potato tuber-specific protein,a Bacillus thuringiensis insecticidal protein, a Xenorhabdus ehlersiiinsecticidal protein, a Photorhabdus insecticidal protein, a Bacilluslaterosporus insecticidal protein or a Bacillus sphaericus insecticidalprotein.
 18. A method for producing a plant capable of controlling aninsect pest of Monolepta hieroglyphica, comprising introducing one ofthe following into the plant: a polynucleotide, wherein thepolynucleotide comprises a polynucleotide sequence that is at least 99%identical to a fragment of SEQ ID NO:1 that is at least 121 nucleotidesin length; the expression cassette comprising the polynucleotide; therecombinant vector comprising the polynucleotide, or an interferingribonucleic acid; wherein the interfering ribonucleic acid comprises atleast one silencing element, wherein the silencing element is adouble-stranded RNA region comprising complementary strands which havebeen annealed, and one strand of which comprises a nucleotide sequenceat least partially complementary to a target sequence within the targetgene, and the target sequence comprises a polynucleotide sequence thatis at least 99% identical to a fragment of SEQ ID NO:1 that is at least121 nucleotides in length.
 19. A method for controlling invasion of aninsect pest of Monolepta hieroglyphica or protecting a plant from damagecaused by an insect pest of Monolepta hieroglyphica, comprisingintroducing one of the following into the plant: the polynucleotide,wherein the polynucleotide comprises a polynucleotide sequence that isat least 99% identical to a fragment of SEQ ID NO:1 that is at least 121nucleotides in length; the expression cassette comprising thepolynucleotide; the recombinant vector comprising the polynucleotide; oran interfering ribonucleic acid; wherein when ingested by the insectpest of Monolepta hieroglyphica, the plant being introduced acts toinhibit growth of the insect pest of Monolepta hieroglyphica; whereinthe interfering ribonucleic acid comprises at least one silencingelement, wherein the silencing element is a double-stranded RNA regioncomprising complementary strands which have been annealed, and onestrand of which comprises a nucleotide sequence at least partiallycomplementary to a target sequence within the target gene, and thetarget sequence comprises a polynucleotide sequence that is at least 99%identical to a fragment of SEQ ID NO:1 that is at least 121 nucleotidesin length.
 20. The isolated polynucleotide according to claim 1, whereinthe polynucleotide sequence is as shown in SEQ ID NO:
 1. 21. Theisolated polynucleotide according to claim 1, wherein the polynucleotidesequence is selected from any one of polynucleotide sequences as shownin SEQ ID NO: 3 to 6.